Products for transfection have long been a staple of biomedical research, and many are now finding potential applications in clinical medicine. In particular, chemical-type and electroporation products are widely used, owing to their ease of use relative to viral transfection. Despite the commercial success in this segment, efficient transfection of some cell types remains difficult. Many of the cell types that are generally resistant to non-viral transfection are immune cells (lymphocytes, macrophages), which places a bottleneck in the development of cellular therapy and basic science. In this proposal, we seek to validate a unique non-viral mechanism of transfection, with particular emphasis on cells that existing products do poorly with (primary B- and T- cells). Our technology is based on sonoporation, in which transient poration is mediated by the interaction of ultrasound energy and compressible microbubble reagents. Despite some recent progress demonstrating feasibility of sonoporation for treating difficult cells, significant work remains to be done in orer to make this technology broadly useful. We have identified performance milestones pertaining to efficiency and purity that are necessary for a viable commercial product. We have developed a set of tools that enable high- throughput evaluation of multiple procedural parameters (concentration, acoustic intensity, pulse length, repetition frequency, microbubble diameter, treatment duration) in a well plate-based assay, and describe an optimization path designed to find treatment conditions under which high transfection efficiency (>70%) and cell viability (>70%) are achieved. We plan to develop a product set consisting of a sonoporation device and cell type-specific microbubble reagents that enables isolation of desired cells from spleen or blood and subsequent transfection using a rapid and robust procedure. We will first evaluate delivery efficiency and viability in a panel of cultured lymphocyte cell lines, with the goal of reducing the parameter space before testing on primary cells. We will then evaluate transfection efficiency for model RNA and DNA payloads in primary mouse T- and B-cells. We will complete our proof-of-concept evaluation by investigating the potential for functional alterations in primar cells transfected using our products. Success in the aims described here will result in data that enables us to begin prototyping a customer-ready sonoporation device and to begin commercialization discussions with potential distribution partners. Downstream applications in clinical cell therapy are anticipated to be the topic for a phase II proposal.